2-substituted-1-deaza purine derivatives with adenosine receptor modulating activity

ABSTRACT

The present invention relates to 2-substituted-1-deaza purine derivatives as adenosine receptor modulating agents, to methods for the preparation of these compounds and to novel intermediates useful for the synthesis of said purine derivatives. 
     The compounds have the general formula (1) 
                         
wherein the symbols have the meanings given in the specification.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/608,145, filed Sep. 9, 2004, the content of which is incorporatedherein by reference.

The present invention relates to 2-substituted-1-deaza purinederivatives as adenosine receptor modulating agents, to methods for thepreparation of these compounds and to novel intermediates useful for thesynthesis of said purine derivatives. The invention also relates to theuse of a compound disclosed herein for the manufacture of a medicamentgiving a beneficial effect. A beneficial effect is disclosed herein orapparent to a person skilled in the art from the specification andgeneral knowledge in the art. The invention also relates to the use of acompound of the invention for the manufacture of a medicament fortreating or preventing a disease or condition. More particularly, theinvention relates to a new use for the treatment of a disease orcondition disclosed herein or apparent to a person skilled in the artfrom the specification and general knowledge in the art. In embodimentsof the invention specific compounds disclosed herein are used for themanufacture of a medicament useful in the treatment of disorders inwhich adenosine receptors are involved, or that can be treated viamanipulation of those receptors.

At present, four types of adenosine receptors have been identified anddesignated A₁, A_(2A), A_(2B) and A₃ respectively. All four belong tothe super-family of seven trans-membrane G-protein coupled receptors.Adenosine receptors are ubiquitous and involved in a great variety ofbiological processes. Thus, during the past decades the therapeuticpotential of adenosine receptor ligands has resulted in a substantialresearch interest.

1-Deaza adenosines are known from EP 0 354 180 A, and were alsodescribed by J. E. Francis et al. (Canadian J. Chem. 70, 1288-1295,1992) and M. J. Wanner et al. (Bioorganic & Medicinal Chemistry Letters2000, 10, 2141-2144). These compounds were shown to have affinity foradenosine receptors, notably for the adenosine A₂ receptor subtype, onwhich they are agonists. The 1-deaza adenosines described in thedocuments cited above all contain the 9-β-D-ribofuranosyl substituentcharacteristic for the natural ligand adenosine. However, highlyhydrophilic moieties like ribose have a negative impact on thebioavailability of compounds bearing such substituents.

The goal of the present invention was to develop 1-deazapurines withouta ribose moiety, but with adenosine receptor modulating activity

Surprisingly it was found that compounds of the general formula (1):

wherein

-   -   R₁ represents oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl,        4-hydroxy-cyclohexyl-NH—, optionally substituted        arylalkyl(C₁₋₃)amines, cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines,        arylalkyl(C₁₋₃)hydrazines or        cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)hydrazines    -   R₂ represents amino, NH-alkyl(C₁₋₃) or N-dialkyl(C₁₋₃)        optionally substituted arylalkyl(C₁₋₃)amines or        cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines,    -   R₃ represents hydrogen, alkyl(C₁₋₃) or arylalkyl(C₁₋₃) and        tautomers, stereoisomers, prodrugs and salts thereof,        are new and are adenosine receptor modulators

Throughout this specification the generally accepted atom numberingsystem of the purine skeleton is used:

The invention relates to racemates, mixtures of diastereomers as well asthe individual stereoisomers of the compounds having formula (1). Theinvention also relates to the E isomer, Z isomer and E/Z mixtures ofcompounds having formula (1). In the description of the substituents theabbreviation ‘alkyl(C₁₋₃)’ means ‘methyl, ethyl, n-propyl or isopropyl’.‘Optionally substituted’ means that a group may or may not be furthersubstituted by one or more groups selected from alkyl, alkenyl, alkynyl,aryl, fluoro, chloro, bromo, hydroxyl, alkyloxy, alkenyloxy, aryloxy,acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio,arylthio, cyano, oxo, nitro, acyl, amido, alkylamido, dialkylamido,carboxyl, or two optional substituents may together with the carbonatoms to which they are attached form a 5- or 6-membered aromatic ornon-aromatic ring containing 0, 1 or 2 heteroatoms selected fromnitrogen, oxygen or sulphur. Within the context of the explanation of‘optionally substituted’, ‘alkyl’ means C₁₋₃-alkyl, ‘alkenyl’ meansC₁₋₃-alkenyl, ‘alkynyl’ means C₁₋₃-alkynyl, ‘acyl’ means C₁₋₃-acyl and‘aryl’ means furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,pyrazolyl, isoxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, 1,3,5-triazynyl, phenyl, indazolyl, indolyl, indolizinyl,isoindolyl, benzi[b]furanyl, benzo[b]thiophenyl, benzimidazolyl,benzthiazolyl, purinyl, quinolynyl, isochinolyl, chinolyl, phtalazinyl,quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, naphthyl orazulenyl, preferably phenyl, pyridyl of naphthyl. Optional substituentsmay themselves bear additional optional substituents. Preferred optionalsubstituents include C₁₋₃ alkyl such as for example methyl, ethyl, andtrifluoromethyl, fluoro, chloro, bromo, hydroxyl, C₁₋₃ alkyloxy such asfor example methoxy, ethoxy and trifluoromethoxy, and amino.

Prodrugs of the compounds mentioned above are in the scope of thepresent invention.

Prodrugs are therapeutic agents which are inactive per se but aretransformed into one or more active metabolites. Prodrugs arebioreversible derivatives of drug molecules used to overcome somebarriers to the utility of the parent drug molecule. These barriersinclude, but are not limited to, solubility, permeability, stability,presystemic metabolism and targeting limitations (Medicinal Chemistry:Principles and Practice, 1994, ISBN 0-85186-494-5, Ed.: F. D. King, p.215; J. Stella, “Prodrugs as therapeutics”, Expert Opin. Ther. Patents,14(3), 277-280, 2004; P. Ettmayer et al., “Lessons learned from marketedand investigational prodrugs”, J.Med.Chem., 47, 2393-2404, 2004).

Pro-drugs, i.e. compounds which when administered to humans by any knownroute, are metabolised to compounds having formula (1), belong to theinvention. In particular this relates to compounds with primary orsecondary amino or hydroxy groups. Such compounds can be reacted withorganic acids to yield compounds having formula (1) wherein anadditional group is present which is easily removed afteradministration, for instance, but not limited to amidine, enamine, aMannich base, a hydroxyl-methylene derivative, an O-(acyloxymethylenecarbamate) derivative, carbamate, ester, amide or enaminone.

The invention particularly relates to compounds of the general formula(1) in which:

-   -   R₁ represents oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl,        4-hydroxy-cyclohexyl-NH—, Ph-CH₂—CH═N—NH—, Ph-CH═N—NH—,        4-methoxy-Ph-CH═N—NH—, 2,4-dimethoxy-Ph-CH═N—NH—,        cyclohexyl-CH₂—N═N— or cyclohexyl-CH═N—NH—,    -   R₂ represents amino and R₃ represents methyl        General Aspects of Syntheses

The synthesis of compounds having formula (I) is outlined in Scheme 1.Modification and functionalization of the 2-position of the regular(1-aza)-purine ringsystem has received much attention. Substitution ofpurine-2-halides with amines proceeds often sluggish and requires hightemperatures. The functionalisation of the 2-position in 1-deazapurinesis even more difficult because it is a very deactivated system. Aneffective alternative is to start with 6-NO₂— or 6-Cl-substituted1-deaza-purines. The synthesis of these starting materials is describedby Cristalli et al. (J. Med. Chem. 1987, 30, 1686-1688) for X=NO₂, andby Itoh et al (J. Heterocyclic Chem. 1982, 19, 513-517) for X=Cl.

After BOC-protection selective nitration on the 2-position can beaccomplished in an analogous way to the nitration of 9-ribose containing1-deazapurines published by Deghati, Bieraugel, Wanner and Koomen (Mildand regioselective nitration of 1-deazapurine nucleosides usingTBAN/TFAA. Tetrahedron Letters 2000, 41, 569-573). After a subsequentmulti-step introduction sequence of a 2-nitroso moiety, the nitrosogroup can be further modified using Diels-Alder, ene reactions, additionreactions, Mills coupling, and condensation reactions. Additionalinformation about the potential further modifications for analogous2-nitrosoadenosines was published by Wanner and Koomen (Synthesis andproperties of 2-nitrosoadenosine. Journal of the Chemical Society-PerkinTransactions 1, 2001, 1908-1915).

The selection of the particular synthetic procedures depends on factorsknown to those skilled in the art such as the compatibility offunctional groups with the reagents used, the possibility to useprotecting groups, catalysts, activating and coupling reagents and theultimate structural features present in the final compound beingprepared.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by mixing a compound ofthe present invention with a suitable acid, for instance an inorganicacid such as hydrochloric acid, or with an organic acid.

The compounds of the invention of the general formula (1), as well asthe salts thereof, have adenosine receptor modulating activity. They areuseful in the treatment of disorders in which adenosine receptors areinvolved, or that can be treated via manipulation of those receptors.For instance in: acute and chronic pain, inflammatory diseasesincluding, arthritis, multiple sclerosis, asthma and psoriasis;gastro-intestinal disorders such as ulcers, inflammatory bowel disease(Crohn's disease) and ulcerative colitis; allergic responses such aseczema, atopic dermatitis and rhinitis; cardio-vascular disorders suchas myocardial infarction, arrhythmias, hypertension, thrombosis,anaemia, arteriosclerosis, angina pectoris, cutaneous diseases such asurticaria, lupus erythematosus and pruritus; opthalmological disorderslike glaucoma; respiratory disorders including chronic obstructivepulmonary disease, bronchitis and cystic fibrosis; central nervoussystem disorders including various forms of epilepsy, stroke,depression, sleep apnoea; disorders characterized by impairment ofcognition and memory such as Alzheimer's disease, Creutzfeldt-Jacobdisease, Huntington's disease, Parkinson's disease, neurorehabilitation(post-traumatic brain lesions); acute brain or spinal cord injury;diabetes, osteoporosis, diseases of the immune system, variouscarcinomas and leukemia, bacterial and viral infections.

Pharmaceutical Preparations

The compounds of the invention can be brought into forms suitable foradministration by means of usual processes using auxiliary substancessuch as liquid or solid carrier material. The pharmaceuticalcompositions of the invention may be administered enterally, orally,parenterally (intramuscularly or intravenously), rectally or locally(topically). They can be administered in the form of solutions, powders,tablets, capsules (including microcapsules), ointments (creams or gel)or suppositories. Suitable excipients for such formulations are thepharmaceutically customary liquid or solid fillers and extenders,solvents, emulsifiers, lubricants, flavorings, colorings and/or buffersubstances. Frequently used auxiliary substances which may be mentionedare magnesium carbonate, titanium dioxide, lactose, mannitol and othersugars or sugar alcohols, talc, lactoprotein, gelatin, starch, celluloseand its derivatives, animal and vegetable oils such as fish liver oil,sunflower, groundnut or sesame oil, polyethylene glycol and solventssuch as, for example, sterile water and mono- or polyhydric alcoholssuch as glycerol.

Compounds of the present invention are generally administered aspharmaceutical compositions which are important and novel embodiments ofthe invention because of the presence of the compounds, moreparticularly specific compounds disclosed herein. Types ofpharmaceutical compositions that may be used include but are not limitedto tablets, chewable tablets, capsules, solutions, parenteral solutions,suppositories, suspensions, and other types disclosed herein or apparentto a person skilled in the art from the specification and generalknowledge in the art. The invention also includes the preparation ormanufacture of said pharmaceutical compositions.

In embodiments of the invention, a pharmaceutical pack or kit isprovided comprising one or more containers filled with one or more ofthe ingredients of a pharmaceutical composition of the invention.Associated with such container(s) can be various written materials suchas instructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals products, which notice reflects approval by the agencyof manufacture, use, or sale for human or veterinary administration.

Pharmacological Methods

In vitro Affinity for Human Adenosine-A₁ Receptors

Affinity of the compounds for human adenosine-A₁ receptors wasdetermined using the receptor binding assay described by A.Townsend-Nicholson and P. R. Schofield (Biol. Chem., 269, 2373, 1994),using human recombinant receptors expressed in CHO cells, and [³H]DPCPXas radioligand.

In vitro Affinity for Human Adenosine-A_(2A) Receptors

Affinity of the compounds for human adenosine-A_(2A) receptors wasdetermined using the receptor binding assay described by D. R. Luthin etal. (Mol. Pharmacol., 47, 307, 1995), using human recombinant receptorsexpressed in HEK-293 cells, and [³H]CGS 21680 as radioligand.

In vitro Affinity for Human Adenosine-A_(2B) Receptors

Affinity of the compounds for human adenosine-A_(2B) receptors wasdetermined using the receptor binding assay described by J. H. Stehle etal. (Mol. Endocrinol., 6, 384, 1992), using human recombinant receptorsexpressed in HEK-293 cells, and [³H]MRS 1754 as radioligand.

In vitro Affinity for Human Adenosine-A₃ Receptors

Affinity of the compounds for human adenosine-A₃ receptors wasdetermined using the receptor binding assay described by C. A. Salvatoreet al.: “Molecular cloning and characterization of the human A ₃adenosine receptor”, Proc. Natl. Acad. Sci. USA, 90, 10365-10369, 1993.Briefly, membrane preparations were obtained from human recombinant (HEK293) cells in which the human adenosine-A₃ receptor was stablyexpressed. Membranes were incubated at 22° C. for 90 minutes with[¹²⁵I]-AB-MECA in the absence or presence of test compounds in aconcentration range from 10 μM down to 0.1 nM, diluted in a suitablebuffer. Separation of bound radioactivity from free was done byfiltration through Packard GF/B glass fiber filters with severalwashings with ice-cold buffer using a Packard cell harvester. Boundradioactivity was measured with a scintillation counter (Topcount,Packard) using a liquid scintillation cocktail (Microscint 0, Packard).Measured radioactivity was plotted against the concentration of thedisplacing test compound and displacement curves were calculated byfour-parameter logistic regression, resulting in IC₅₀ values, i.e. thatconcentration of displacing compound by which 50% of the radioligand isdisplaced. Affinity pK_(I) values were calculated by correcting the IC₅₀values for radioligand concentration and its affinity for the humanadenosine-A₃ receptor according to the Cheng-Prusoff equation:pK _(I)=−log (IC ₅₀/(1+S/K _(d)))in which the IC₅₀ is as described above, S is the concentration[¹²⁵I]-AB-MECA used in the assay expressed in mol/l (typically 0.1 nM),and K_(d) is the equilibrium dissociation constant of [¹²⁵I]-AB-MECA forhuman adenosine-A₃ receptors (0.22 nM).

The compounds of the invention have affinity for at least one of theadenosine receptors in one of the binding assays described above. Theseproperties make them useful in the treatment of disorders in whichadenosine receptors are involved, or that can be treated viamanipulation of these receptors.

According to the procedures described above the compounds of theinvention can be prepared. The examples are intended to furtherillustrate the invention in more detail, and therefore are not deemed torestrict the scope of the invention in any way.

Dose

The affinity of the compounds of the invention for adenosine A₃receptors was determined as described above. From the binding affinitymeasured for a given compound of formula (1), one can estimate atheoretical lowest effective dose. At a concentration of the compoundequal to twice the measured K_(i)-value, nearly 100% of the adenosine A₃receptors likely will be occupied by the compound. Converting thatconcentration to mg of compound per kg of patient yields a theoreticallowest effective dose, assuming ideal bioavailability. Pharmacokinetic,pharmacodynamic, and other considerations may alter the dose actuallyadministered to a higher or lower value. The dosage expedientlyadministered is 0.001-1000 mg/kg, preferably 0.1-100 mg/kg of patient'sbodyweight.

Treatment

The term “treatment” as used herein refers to any treatment of a humancondition or disease and includes: (1) preventing the disease orcondition from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it, (2) inhibiting thedisease or condition, i.e., arresting its development, (3) relieving thedisease or condition, i.e., causing regression of the condition, or (4)relieving the conditions caused by the disease, i.e., stopping thesymptoms of the disease.

EXAMPLES Example 1 Materials and Methods

All reactions involving moisture sensitive compounds were carried outunder a dry nitrogen atmosphere. Dichloromethane (phosphorous pentoxideand calciumhydride), tetrahydrofuran (sodium/benzophenone ketyl) andlight petroleum (60-80) were distilled freshly prior to use. All othercommercially available chemicals were used without further purification.Reactions were monitored by using thin-layer chromatography (TLC) onsilica coated plastic sheets (Merck silica gel 60 F254) with theindicated eluent. The compounds were visualised by UV light (254 nm) orI₂. Flash chromatography refers to purification using the indicatedeluent and Acros silica gel (0.030-0.075 mm). Nuclear magnetic resonancespectra (¹H NMR and ¹³C NMR, APT) were determined in the indicatedsolvent using a Bruker ARX 400 (¹H: 400 MHz, ¹³C: 100 MHz) at 300 K,unless indicated otherwise. ¹⁹F NMR and ¹³C NMR experiments were carriedout on a Varian Inova 500 spectrometer operating at 11.74 T (499.9 MHzfor ¹H; 125.7 MHz for ¹³C; 50.7 Mhz, 470.4 MHz for ¹⁹F) using a 5 mm SWprobe. The spectra were determined in deuterated chloroform ordichloromethane obtained from Cambridge Isotope Laboratories Ltd.Chemical shifts (δ) are given in ppm downfield from tetramethylsilane(1H, 13C) or CCl3F (¹⁹F). Coupling constants J are given in Hz.Peakshapes in the NMR spectra are indicated with the symbols ‘q’(quartet), ‘dq’ (double quartet), ‘t’ (triplet), ‘dt’ (double triplet),‘d’ (doublet), ‘dd’ (double doublet), ‘s’ (singlet), ‘bs’ (broadsinglet) and ‘m’ (multiplet). NH and OH signals were identified aftermixing the sample with a drop of D₂O. Melting points were measured witha Büchi B-545 Melting Point apparatus. Mass spectra and accurate massmeasurements were performed using a JEOL JMS-SX/SX 102 A Tandem MassSpectrometer using Fast Atom Bombardement (FAB). A resolving power of10,000 (10% valley definition) for high resolution FAB mass spectrometrywas used. Extinction coefficients were determined with a HP 8453 UV-Visspectrophotometer. Analytical HPLC was performed on a C18 column(Inertsil ODS-3, particle size 3 mm; 4.6 mm 50 mm) using the followingelution gradient: linear gradient of 5% to 95% aqueous CH₃CN containing0.04% HCO₂H over 5 min, then 95% aqueous CH₃CN containing 0.04% HCO₂Hfor 2 min at 2.0 ml min⁻¹. Products were detected at λ=254 nm.

Example 2 Syntheses of Intermediates Intermediate A:6-Chloro-9-Boc-1-deazapurine

To a solution of 6-chloro-1-deazapurine (10 g, 65.1 mmol, J.Heterocyclic Chem. 1982, 19, 513-517) and BOC₂O (19.8 g, 91.2 mmol) indry dichloromethane (100 ml) was added dimethylaminopyridine (0.5 g, 5mass %) and the mixture was stirred at room temperature for 30 minutes.The reaction was quenched by adding silica and the mixture was filteredover hyflo. Evaporating the solvent yielded the crude product.Trituration with petroleum ether followed by treatment with etheryielded the product as a white solid (6.29 g, 76%), mp: 183.5° C.decomposition

¹H NMR (d₆-DMSO) δ 8.89 (s, 1H, H-8), 8.47-8.46 (d, J 5.3 , 1H, H-2),7.62-7.61 (1H, d, J 5.3, H-1), 1.66 ( s, 9H t-Bu).

Intermediate B: 2-nitro-6-chloro-9-Boc-1-deazapurine

A solution of 6-chloro-9-Boc-1-deazapurine (1 g, 3.94 mmol, intermediateA) and tetrabutyl-ammonium nitrate (1.8 g, 5.91 mmol) in drydichloromethane (12 ml) was stirred in an ice bath. TFAA (835 μL, 5.91mmol) was added dropwise. After 1.5 h the reaction was complete and MeOH(20 ml) was added. The mixture was concentrated and cooled to giveyellow crystals. The crude product was washed with 3×3 ml cold MeOH.Drying in vacuo at 50° C. afforded the product (0.916 g, 78%) as yellowneedles. mp: 288.2-290° C. decomposition ¹H NMR (d₆-DMSO) δ 9.22 (s,1H,H-8), 8.58 (s, 1H, H-1), 1.68 (s, 9H, t-Bu).

Intermediate C: 2-nitro-6-chloro-1-deazapurine

2-nitro-6-chloro-9-Boc-1-deazapurine (1.0 g, 3.35 mmol, intermediate B)in MeOH (20 ml) was stirred at 70° C. After 2 h the product started toprecipitate. After 15 h the suspension was cooled at 0° C. for 30 minand filtered. The precipitate was washed with cold MeOH and dried invacuo at 50° C. to furnish the product (0.5 g, 76%) as a yellow solid.mp: 294.5° C.

¹H NMR(d₆-DMSO) δ 14.20 (bs, 1H, H-9), 8.92 (s, 1H, H-8), 8.41 (d, 1H,H-1).

Intermediate D: 6-nitro-9-Boc-1-deazapurine

To a solution of 6-nitro-1-deazapurine (5 g, 30 mmol, J.Med.Chem., 1987,30, 1686-1688) and BOC₂O (10 g, 46 mmol) in dry dichloromethane (100 ml)was added dimethylaminopyridine (0.250 g, 5 mass %) and the mixture wasstirred at room temperature for 1.5 h. The reaction was diluted with PEand quenched by adding silica and the mixture was filtered over hyflo.Evaporating the solvent yielded the crude product. Crystallization withEA/PE afforded the product (2.28 g, 72%) as light yellow needles. mp:223-226° C.

¹H NMR (d₆-DMSO) δ 9.11 (s, 1H, H-8), 8.81 (d, J 5.3 , 1H, H-2), 8.10 (dJ 5.3, 1H, H-1), 1.67 ( s, 9H t-Bu).

Intermediate E: 2,6-dinitro-1-deazapurine

A solution of 6-nitro-9-Boc-1-deazapurine (4 g, 15 mmol, intermediate D)and tetrabutylammoniumnitrate (0.92 g, 23 mmol) in dry dichloromethane(5 ml) was stirred at −18° C. TFAA (430 μL, 18 mmol) was added dropwise.After 1.5 h the nitration reaction was complete and MeOH (20 ml) wasadded. The solution was refluxed for 15 h while the productprecipitates. Cooling and filtering and drying the suspension furnishedthe pure product (2.28 g, 72%) as light yellow crystals. mp: 294° C.

¹H NMR (d₆-DMSO) δ 14.45(bs, 1H, H-9), 9.16(s, 1H, H-8), 8.81 (d, 1H,H-1).

Intermediate F: 2-nitro-6-azido-1-deazapurine

First possible route: a suspension of 2-nitro-6-chloro-1-deazapurine (12mmol, intermediate C) and NaN₃ (2.5 g, 37 mmol) in dry DMF (25 ml) wasstirred and warmed to 60° C. After 60 h the reaction was cooled to roomtemperature and water was added slowly (100 ml). The productprecipitated and was filtered. Washing with water and ether, followed bydrying in vacuo at 50° C. furnished the product (2.12 g, 82%) as asolid.

Alternative route: a suspension of 2,6-dinitro-1-deazapurine (2.5 g, 12mmol, intermediate E) and NaN₃ (2.4 g, 37 mmol) in dry DMF (20 ml) wasstirred at room temperature for 3 h. After completion, water was addedslowly (100 ml). The product precipitated and was filtered. Washing withwater and ether, followed by drying in vacuo at 50° C. furnished theproduct (2.22 g, 90%) as a off white solid. mp: 220-224 ° C.decomposition

¹H NMR(d₆-DMSO) δ 14.02 (bs, 1H, H-9), 8.78 (s, 1H, H-8), 7.81 (s, 1H,1-H).

Intermediate G: 2-nitro-6-azido-9-methyl-1-deazapurine

To a suspension of 2-nitro-6-azido-1-deazapurine (6 g, 29.2 mmol,Intermediate F) and K₂CO₃ (8.09 g, 58.5 mmol) in dry DMF (150 ml) wasadded Mel (3.65 ml, 58.5 mmol). After 1 h the reaction was complete andwater (300 ml) was added slowly. The product precipitated and thesuspension was cooled in an ice-bath. The crude product was washed withwater and ether and dried in vacuo at 50° C. to yield 5.61 g (88%) ofthe desired product as a white solid. mp: 171.7° C. decomposition

¹H NMR (d₆-DMSO) δ 8.77 (s, 1H H-8), 7.80 (s, 1H, H-1), 3.92 (s, 3H,CH₃).

Intermediate H: 2-nitro-6-amino-9-Me-1-deazapurine

To a solution of 2-nitro-6-azido-9-methyl-1-deazapurine (1 g, 4.5 mmol,Intermediate G) was added Pd/C 10% (0.200 g, 20 mol %). The solution wasstirred at room temperature under a H₂ atmosphere. After 1 h thereaction was complete. Flash chromatography (EA) of the crude reactionmixture afforded the pure product as a red solid (0.775 g. 88%). mp:260-264° C.

¹H NMR (d₆-DMSO) δ 8.35 (s, 1H H-8), 7.36 (s, 1H, H-1), 7.15 (bs, 2H,NH₂), 3.79 (s, 3H, CH₃).

Intermediate I: 2-hydroxylamino-6-amino-9-methyl-1-deazapurine

A suspension of 2-nitro-6-azido-9-methyl-1-deazapurine (1 g, 45.6 mmol,Intermediate H) and 10% Pt/C (0.2 g, 20% m) in EA (200 ml) was refluxedunder a H₂ athmosphere. After 18 h the reaction was filtered over Hyflo(EA/MeOH). The filtrate was used immediately for the oxidation reactionto the nitroso derivative.

¹H NMR (d₆-DMSO) δ 8.34 (s, 1H, H-8), 7.36 (s, 1H, H-1), 7.14 (bs, 2H,NH₂), 3.80 (s, 3H, CH₃).

Intermediate J: 2-nitroso-6-amino-9-methyl-1-deazapurine

The crude mixture of 2-hydroxylamino-6-amino-9-methyl-1-deazapurine(intermediate I) was cooled to 0° C. An ice-cold solution of NaIO₄ (1.95g, 9.13 mmol) in H₂O (50 ml) was added slowly. After 1 h the reactionwas complete and the organic layer was separated from the water layer.The aqueous layer was extracted with EA+5% MeOH and dried with Na₂SO₄.The yellow solution was concentrated and triturated with cold MeOH toyield 0.53 g (65%) of the desired product as a red solid. mp: 220° C.

¹H NMR (d₆-DMSO) δ 8.46 (s, 1H, H-8), 7.37 (s, 1H, H-1), 7.00 (bs, 2H,NH₂), 3.92 (s, 3H, CH₃)

Example 3 Syntheses of Specific Compounds Compound 1:2-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-6-amino-9-methyl-1-deazapurine

To a suspension of 2-nitroso-6-amino-9-methyl-1-deazapurine (0.1 g, 0.56mmol, intermediate J) in MeOH (15 ml) was added slowly1,3-cyclohexadiene (105 μl, 1.13 mmol). After 30 m the reaction wascomplete and the mixture was concentrated and triturated in cold MeOH.The product was washed with cold MeOH and dried at 50° C. in vacuo toyield 101.9 mg (75%) of the desired product as a white solid. mp: 169.9°C.

¹H NMR (d₆-DMSO) δ 7.82 (s, 1H, H-8), 6.52 (t, 1H, CH), 6.30 (t, 1H,CH), 6.12 (bs, 2H, NH₂), 5.99 (s, 1H, H-1), 5.21 (t, 1H, CH), 4.70 (t,1H, CH), 3.63 (s, 3H, CH₃), 2.08 (q, 2H, CH₂), 1.55 (q, 2H, CH₂), 1.33(q, 2H, CH₂); m/z 258.1355 (M⁺+H. C₁₃H₁₆N₅O requires m/z 258.1277)

Compound 2:2-(cis-4-hydroxycyclohexylamino)-6-amino-9-methyl-1-deazapurine

A suspension of2-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-6-amino-9-methyl-1-deazapurine(50 mg, 0.194 mmol, compound 1) and 10% Pd/C in MeOH (4 ml) was stirredunder 1 atm hydrogen for 1.5 h at 70 ° C. Filtration, evaporation andstirring in cold MeOH afforded the pure2-(cis-4-hydroxycyclohexylamino)-6-amino-9-methyl-1-deazapurine (23,8mg, 47%) as white solid.

¹H NMR (d₆-DMSO) δ 7.62 (s, 1H, 8-H), 5.72 (s, 2H, NH₂), 5.69-5.67 (m,1H, NH), 5.57 (s, 1h, 1-H), 4.34 (s, 1H, OH), 3.69 (s, 2H), 3.57 (s, 3H,CH₃), 1.63 (m, 6H, cyclo), 1.53-151 (m, 2H, cyclo) m/z 262.1668 (M⁺+H.C₁₃H₁₉N₅O requires m/z 262.1590)

Compound 3:2-(2-phenylethyl-hydrazone)-6-amino-9-methyl-1-deazapurine

A mixture of 2-nitroso-6-amino-9-methyl-1-deazapurine (0.1 g, 0.565mmol), 2-phenylethylamine (858 μl, 0.677 mmol) and acetic acid CH₃COOH(64.6 μl, 1.13 mmol) was stirred in MeOH. After 3 h the suspension haddissolved. The yellow solution was concentrated and diluted with PE andpurified by flash chromatography (EA/MeOH/NH₄OH 88.5:10:1.5). Theproduct was triturated with cold methanol and dried in vacuo to yield55.4 mg (35%) of the desired product as a yellow solid. mp: 182.1° C.

¹H NMR (d₆-DMSO) δ 10.07 (s, 1H, NH), 7.73 (s, 1H, 8-H), 7.37-7.32 (3H,m, CH/Ph), 7.27-7.25-7.23 (m, 3H, Ph), 6.28 (s, 1H, 1-H), 6.1 (bs, 2H,NH₂), 3.60 (s, 3H, CH₃), 3.56-3.54 (d, 2H, CH₂) m/z 281.1515 (M⁺+H.C₁₃H₁₉N₅O requires m/z 281.1436)

Compound 4: 2-benzylhydrazone-6-amino-9-methyl-1-deazapurine

Condensation of 2-nitroso-6-amino-9-methyl-1-deazapurine (0.1 g, 0.565mmol), benzylamine (740 μl, 6.77 mmol) and acetic acid CH₃COOH (64.6 μl,1.13 mmol) was performed as described for compound 3. Flashchromatography (EA/MeOH/NH₄OH 88.5:10:1.5). The product was trituratedwith cold methanol and dried in vacuo to yield 52.6 mg (35%) of thedesired product as a yellow solid. mp: 231.7-232.8° C.;

¹H NMR (d₆-DMSO) δ 10.68 (s, 1H, NH), 7.95 (s, 1H, 8-H), 7.77 (s, 1H,CH), 7.64-7.62 (d, 2H, o-Ph), 7.41 (t, 2H, m-Ph), 7.31 (t, 1H, p-Ph),6.46 (s, 1H, 1-H), 6.17 (bs, 2H, NH₂) m/z 267.1358 (M⁺+H. C₁₃H₁₉N₅Orequires m/z 267.1280)

Compound 5:2-(4-methoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine

Condensation of 2-nitroso-6-amino-9-methyl-1-deazapurine (0.1 g, 0.565mmol), 4-methoxy-benzylamine (369 μl, 2.82 mmol) and acetic acid CH₃COOH(64.6 μl, 1.13 mmol) was performed as described for compound 3. Flashchromatography (EA/MeOH/NH₄OH 94.5:5:0.5). The product was trituratedwith cold methanol and dried in vacuo to yield 58.5 mg (35%) of thedesired product as a yellow solid.

¹H NMR (d₆-DMSO) δ 10.39 (s, 1H, NH), 7.91 (s, 1H, 8-H), 7.75 (s, 1H,CH), 7.56 (d, J 8.53 Hz, 2H, m-Ph), 6.98 (d, J 8.52 Hz, 2H, o-Ph), 6.42(s, 1H, 1-H), 6.13 (bs, 2H, NH₂), 3.80 (s, 3H, OCH₃), 3.64 (s, 3H, CH₃)m/z 297.1464. (M⁺+H. C₁₃H₁₉N₅O requires m/z 297.3271

Compound 6:2-(2,4-dimethoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine

Condensation of 2-nitroso-6-amino-9-methyl-1-deazapurine (0.075 g, 0.423mmol), 2,4-dimethoxy-benzylamine (318 μl, 2.12 mmol) and acetic acidCH₃COOH (48.4 μl, 0.847 mmol) was performed as described for compound 3.Flash chromatography (EA/MeOH/NH₄OH 94.5:5:0.5). The product wastriturated with cold methanol and ether and dried in vacuo to yield 48.3mg (35%) of the desired product as a off white solid.

¹H NMR (d₆-DMSO) δ 10.38 (s, 1H, NH), 8.17 (s, 1H, 8-H), 7.78-7.75 (s,2H, CH and 1-H), 6.61-6.6 (m, 2H, Ph), 6.40 (s, 1H, Ph), 6.11 (bs, 2H,NH₂), 3.85 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.63 (s, 3H, CH₃) m/z327.1569 (M⁺+H. C₁₃H₁₉N₅O requires m/z 327.3531

Compound 7: 2-cyclohexylmethylazo-6-amino-9-methyl-1-deazapurine

Condensation of 2-nitroso-6-amino-9-methyl-1-deazapurine (0.1 g, 0.565mmol), cyclohexylmethylamine (367 μl, 2.82 mmol) and acetic acid CH₃COOH(64.6 μl, 1.13 mmol) was performed as described for compound 3. Flashchromatography (EA/MeOH/NH₄OH 94.5:5:0.5). The product was trituratedwith ether and dried in vacuo to yield 72.6 mg (48%) of the desiredproduct as a bright yellow solid.

¹H NMR (d₆-DMSO) δ 9.81 (s, 1H, NH), 7.71 (1H, s, 8-H), 7.17 (d J 5.31Hz, 1H, CH), 6.22 (s, 1H, 1-H), 6.04 (s, 2H, NH₂), 3.6 (s, 3H, CH₃),2.18-2.17 (m, 1H, cyclohexyl), 1.8-1.76-1.71 (m, 5H, 5×CH) 1.29-1.18 (m,5H, CH) m/z 273.1828 (M⁺+H. C₁₃H₁₉N₅O requires m/z 273.1749

Compound 8:2-(cyclohexylmethyl-hydrazone)-6-amino-9-methyl-1-deaza-purine

2-cyclohexylmethylazo-6-amino-9-methyl-1-deazapurine (0.03 g , 0.111mmol, compound 7) was dissolved in EtOH (5 ml) and a catalytic amount ofN(Et)₃ The mixture was refluxed overnight. The mixture was concentratedin vacuo, triturated with cold ether and MeOH and filtered producing 8mg (27%) of the desired product as a yellow solid.

¹H NMR (d₆-DMSO) δ 9.81 (s, 1H, NH), 7.71 (s, 1H, 8-H), 7.16 (d, J 4.91Hz, 1H, CH), 6.22 (s, 1H, 1-H), 6.04 (s, 2H, NH₂), 3.6 (s, 3H, CH₃)

TABLE I Structural variations of compounds 1-8

Compound R₁ R₂ R₃ 1 2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl NH₂ CH₃ 24-hydroxy-cyclohexyl-NH— NH₂ CH₃ 3 Ph—CH₂—CH═N—NH— NH₂ CH₃ 4 Ph—CH═N—NH—NH₂ CH₃ 5 4-methoxy-Ph—CH═N—NH— NH₂ CH₃ 6 2,4-dimethoxy-Ph—CH═N—NH— NH₂CH₃ 7 Cyclohexyl-CH₂—N═N— NH₂ CH₃ 8 Cyclohexyl-CH═N—NH— NH₂ CH₃

Example 4 Formulation of Comp. 7 used in Animal Studies

For oral (p.o.) administration: to the desired quantity (0.5-5 mg) ofthe solid compound 7 in a glass tube, some glass beads were added andthe solid was milled by vortexing for 2 minutes. After addition of 1 mlof a solution of 1% methylcellulose in water and 2% (v/v) of Poloxamer188 (Lutrol F68), the compound was suspended by vortexing for 10minutes. The pH was adjusted to 7 with a few drops of aqueous NaOH(0.1N). Remaining particles in the suspension were further suspended byusing an ultrasonic bath.

For intraperitoneal (i.p.) administration: to the desired quantity(0.5-15 mg) of the solid compound 7 in a glass tube, some glass beadswere added and the solid was milled by vortexing for 2 minutes. Afteraddition of 1 ml of a solution of 1% methylcellulose and 5% mannitol inwater, the compound was suspended by vortexing for 10 minutes. Finallythe pH was adjusted to 7.

Example 5 Pharmacological Test Results

Adenosine receptor affinity data obtained according to the protocolsgiven above are shown in the table below.

TABLE II In vitro adenosine receptor affinity of compounds 1-8 Adenosinereceptor affinity A₁ A_(2A) A₃ Compound pK_(i) pK_(i) pK_(i) 1 —.— 4.8—.— 2 —.— 5.1 —.— 3 5.6 5.5 —.— 4 5.4 5.9 5.6 5 —.— 6.0 —.— 6 5.2 5.7—.— 7 6.0 6.1 —.— 8 5.5 5.7 —.—

1. A compound of formula (1)

or a pharmaceutically acceptable salt, a stereoisomer, a tautomer, or amixture of any of the foregoing wherein: R₁ is chosen from2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl, 4-hydroxy-cyclohexyl-NH—, andoptionally substituted arylalkyl(C₁₋₃)amines,cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines, arylalkyl(C₁₋₃) hydrazines andcycloalkyl(C₃₋₈)alkyl (C₁₋₃)hydrazines; R₂ is chosen from amino,NH-alkyl(C₁₋₃), N-dialkyl(C₁₋₃), and optionally substitutedarylalkyl(C₁₋₃)amines and cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines; and R₃ ischosen from alkyl(C₁₋₃), and arylalkyl(C₁₋₃).
 2. The compound accordingto claim 1, wherein R₁ is chosen from oxa-3-azabicyclo[22.2]oct-5-en-3-yl, 4-hydroxy-cyclohexyl-NH—, Ph-CH₂—CH═N—NH—,Ph-CH═N—NH—, 4-methoxy-Ph-CH═N—NH—, 2,4-dimethoxy-Ph-CH═N—NH—,cyclohexyl-CH₂—N═N— and cyclohexyl-CH═N—NH—; R₂ is an amino group; andR₃ is a methyl group.
 3. The compound according to claim 1, wherein thecompound of formula (1) is chosen from:2-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-6-amino-9-methyl-1-deazapurine;2-(cis-4-hydroxycyclohexylamino)-6-amino-9-methyl-1-deazapurine;2-(2-phenylethyl-hydrazone)-6-amino-9-methyl-1-deazapurine;2-benzylhydrazone-6-amino-9-methyl-1-deazapurine;2-(4-methoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine;2-(2,4-dimethoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine;2-cyclohexylmethylazo-6-amino-9-methyl-1-deazapurine; and2-(cyclohexylmethyl-hydrazone)-6-amino-9-methyl-1-deaza-purine.
 4. Apharmaceutical composition comprising: at least one pharmaceuticallyacceptable component chosen from a carrier, an auxiliary substance andcombinations thereof; and a therapeutically effective amount of at leastone compound of formula (1)

or pharmaceutically acceptable salts, a stereoisomer, a tautomer, or amixture thereof, wherein: R₁ is chosen from2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl, 4-hydroxy-cyclohexyl-NH—, andoptionally substituted arylalkyl(C₁₋₃)amines,cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines, arylalkyl(C₁₋₃) hydrazines andcycloalkyl(C₃₋₈)alkyl(C₁₋₃)hydrazines; R₂ is chosen from amino,NH-alkyl(C₁₋₃), N-dialkyl(C₁₋₃), and optionally substitutedarylalkyl(C₁₋₃)amines and cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines; and R₃ ischosen from alkyl(C₁₋₃), and arylalkyl(C₁₋₃).
 5. The pharmaceuticalcomposition of claim 4, wherein: R₁ is chosen fromoxa-3-azabicyclo[2.2.2]oct-5-en-3-yl, 4-hydroxy-cyclohexyl-NH—,Ph-CH₂—CH═N—NH—, Ph-CH═N—NH—, 4-methoxy-Ph-CH═N—NH—,2,4-dimethoxy-Ph-CH═N—NH—, cyclohexyl-CH₂—N═N— and cyclohexyl-CH═N—NH—;R₂ is an amino group; and R₃ is a methyl group.
 6. The pharmaceuticalcomposition of claim 4, wherein the compound of formula (1) is chosenfrom:2-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-6-amino-9-methyl-1-deazapurine;2-(cis-4-hydroxycyclohexylamino)-6-amino-9-methyl-1-deazapurine;2-(2-phenylethyl-hydrazone)-6-amino-9-methyl-1-deazapurine;2-benzylhydrazone-6-amino-9-methyl-1-deazapurine;2-(4-methoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine;2-(2,4-dimethoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine;2-cyclohexylmethylazo-6-amino-9-methyl-1-deazapurine; and2-(cyclohexylmethyl-hydrazone)-6-amino-9-methyl-1-deaza-purine.
 7. Thecomposition according to claim 4, wherein the at least onepharmaceutically acceptable auxiliary substance is chosen from magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars or sugaralcohols, talc, lactoprotein, gelatin, starch, cellulose and itsderivatives, animal and vegetable oil, polyethylene glycol, mon- andpolyhydric alcohol, and combinations thereof.
 8. The compositionaccording to claim 4, wherein the composition is in a form chosen fromsolutions, powders, tablets, capsules, ointments, and suppositories. 9.A method for preparing a pharmaceutical composition, comprising:combining a pharmacologically active amount of at least one compound offormula (1)

or pharmaceutically acceptable salts, a stereoisomer, a tautomer, or amixture thereof, wherein: R₁ is chosen from2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl, 4-hydroxy-cyclohexyl-NH—, andoptionally substituted arylalkyl(C₁₋₃)amines,cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines, arylalkyl(C₁₋₃) hydrazines andcycloalkyl(C₃₈)alkyl(C₁₋₃)hydrazines; R₂ is chosen from amino,NH-alkyl(C₁₋₃), N-dialkyl(C₁₋₃), and optionally substitutedarylalkyl(C₁₋₃)amines and cycloalkyl(C₃₋₈)alkyl-(C₁₋₃)amines; and R₃ ischosen from alkyl(C₁₋₃), and arylalkyl(C₁₋₃) and at least onepharmaceutically acceptable component chosen from a carrier, anauxiliary substance, and combinations thereof.
 10. The method of claim9, wherein: R₁ is chosen from oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl,4-hydroxy-cyclohexyl-NH—, Ph-CH₂—CH═N—NH—, Ph-CH═N—NH—,4-methoxy-Ph-CH═N—NH—, 2,4-dimethoxy-Ph-CH═N—NH—, cyclohexyl-CH₂—N═N—and cyclohexyl-CH═N—NH—; R₂ is an amino group; and R₃ is a methyl group.11. The method of claim 9, wherein the at least one compound of formula(1) is chosen from:2-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-6-amino-9-methyl-1-deazapurine;2-(cis-4-hydroxycyclohexylamino)-6-amino-9-methyl-1-deazapurine;2-(2-phenylethyl-hydrazone)-6-amino-9-methyl-1-deazapurine;2-benzylhydrazone-6-amino-9-methyl-1-deazapurine;2-(4-methoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine;2-(2,4-dimethoxy-2-benzylhydrazon)-6-amino-9-methyl-1-deazapurine;2-cyclohexylmethylazo-6-amino-9-methyl-1-deazapurine; and2-(cyclohexylmethyl-hydrazone)-6-amino-9-methyl-1-deaza-purine.
 12. Thecomposition according to claim 9, wherein the at least onepharmaceutically acceptable auxiliary substance is chosen from magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars or sugaralcohols, talc, lactoprotein, gelatin, starch, cellulose and itsderivatives, animal and vegetable oil, polyethylene glycol, mon- andpolyhydric alcohol, and combinations thereof.
 13. The method of claim 9,wherein the pharmaceutical composition is in a form chosen fromsolutions, powders, tablets, capsules, ointments, and suppositories.